N.BstNBI is a nicking endonuclease that recognizes the sequence GAGTC and nicks one DNA strand specifically. The Type IIs endonuclease, MlyI, also recognizes GAGTC, but cleaves both DNA strands. Sequence comparisons revealed significant similarities between N.BstNBI and MlyI. Previous studies showed that MlyI dimerizes in the presence of a cognate DNA, whereas N.BstNBI remains a monomer. This suggests that dimerization may be required for double-stranded cleavage. To test this hypothesis, we used a multiple alignment to design mutations to disrupt the dimerization function of MlyI. When Tyr491 and Lys494 were both changed to alanine, the mutated endonuclease, N.MlyI, no longer formed a dimer and cleaved only one DNA strand specifically. Thus, we have shown that changing the oligomerization state of an enzyme changes its enzymatic function. This experiment also established a protocol that could be applied to other Type IIs endonucleases in order to generate more novel nicking endonucleases.